Screening for Proteases Specifically Hydrolyzing IgE Epitopes aa 83-105 of α_s1-Casein Allergen

The aim of this study was to explore a method for screening for proteases specifically hydrolyzing the epitopes of α_s1-casein. First, the epitope aa 83–105 of α_s1-casein was synthesized by solid-phase synthesis method. After purification and identification, the peptide was coupled to bovine serum albumin (BSA) to prepare a complete antigen, and then BALB/c mice were immunized to prepare monoclonal antibodies. In addition, an indirect competitive enzyme linked immunosorbent assay (ELISA) was established. The monoclonal antibody prepared with α_s1-casein and the established method were regarded as controls. The results showed that the purity of the synthetic epitope was over 90%, and the coupling rate with BSA was 6.31. The monoclonal antibody belonged to IgG₁, with a titer of 1:320000, and it could react specifically with α_s1-casein, but did not cross-react with soybean protein. The linear regression equation of the competitive inhibition curve was y = - 9.22x + 100.78 (R² = 0.9891). The indirect competitive ELISA method showed good repeatability and accuracy, and its detection limit was lower than that of the monoclonal antibody method. The amounts of residual antigen in papain and alcalase hydrolysates were relatively small, which needs to be further verified by in vivo test. The successful preparation of G₁ monoclonal antibody against α_S1-casein epitope aa 83–105 provides a specialized tool for ELISA detection of antigen residues and for the development of hypoallergenic formula.